HIV/AIDS afflicts 33 million people and a preventive vaccine that will stop transmission of HIV is desperately needed. Recombinant BCG (rBCG) and M. smegmatis (rSmeg) are promising vaccine vectors that were shown to induce protective immunity against a number of pathogens in laboratory animals. However, there is ample evidence demonstrating that current mycobacteria vaccine vectors have limitations. rBCG and rSmeg vectors that are immunogenic are unstable since they are transformed with multi-copy and episomal expression plasmids. Those mycobacteria vaccine vectors that are produced by integrative single-copy plasmids are stable but do not express sufficient amounts of the vaccine antigen to induce an immune response in mice and monkeys. In this proposal, we will explore an innovative approach by exploiting mycobacteriophages to generate integration-proficient expression plasmids for engineering second-generation rBCG and rSmeg vaccine vectors that are not only stable but also have markedly higher expression of vaccine antigens. Preclinical immunogenicity testing in mice will be conducted to assess whether the second generation mycobacteria vaccine vectors will outperform old prototypes in inducing anti-SIV immune responses. We will partner the new rBCG and rSmeg vectors with recombinant adenovirus vectors for eliciting robust systemic and mucosal antibody and T cell responses. These preclinical studies are a critical step towards developing new and improved recombinant BCG and M. smegmatis as potential candidate vaccine vectors against AIDS and other diseases.